Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L

The S-1 and S-2 subsite specificity of recombinant human cathepsins X was studied using fluorescence resonance energy transfer (FRET) peptides with the general sequences Abz-Phe-Xaa-Lys(Dnp)-OH and Abz-XaaArg-Lys(Dnp)-OH, respectively (Abz=ortho-aminobenzoic acid and Dnp=2,4-dinitrophenyl; Xaa=various amino acids). Cathepsin X cleaved all substrates exclusively as a carboxymonopeptidase and exhibited broad specificity. for comparison, these peptides were also assayed with cathepsins B and L. Cathepsin L hydrolyzed the majority of them with similar or higher catalytic efficiency than cathepsin X, acting as an endopeptidase mimicking a carboxymonopepticlase (pseudo-carboxymonopeptidase). in contrast, cathepsin B exhibited poor catalytic efficiency with these substrates, acting as a carboxydipeptidase or an endopeptidase. the S-1' subsite of cathepsin X was mapped with the peptide series AbzPhe-Arg-Xaa-OH and the enzyme preferentially hydrolyzed substrates with hydrophobic residues in the P-1' position.Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilJozef Stefan Inst, Dept Biochem & Mol Biol, SI-1000 Ljubljana, SloveniaUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

A possible alternative mechanism of kinin generation in vivo by cathepsin L

We investigated the ability of cathepsin L to induce a hypotensive effect after intravenous injection in rats and correlated this decrease in blood pressure with kinin generation. Simultaneously with blood pressure decrease, we detected plasma kininogen depletion in the treated rats. the effect observed in vivo was abolished by preincubation of cathepsin L with the cysteine peptidase-specific inhibitor E-64 (11 mu m) or by previous administration of the bradykinin B-2 receptor antagonist JE049 (4 mg/kg). A potentiation of the hypotensive effect caused by cathepsin L was observed by previous administration of the angiotensin I-converting enzyme inhibitor captopril (5 mg/kg). in vitro studies indicated that cathepsin L excised bradykinin from the synthetic fluorogenic peptide Abz-MTSVIRRPPGFSPFRAPRV-NH2, based on the Met(375)-Val(393) sequence of rat kininogen (Abz=o-aminobenzoic acid). in conclusion, our data indicate that in vivo cathepsin L releases a kinin-related peptide, and in vitro experiments suggest that the kinin generated is bradykinin. Although it is well known that cysteine proteases are strongly inhibited by kininogen, cathepsin L could represent an alternative pathway for kinin production in pathological processes.UNIFESP, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilUSP, Fac Ciencias Farmaceut Ribeirao Preto, BR-14049900 São Paulo, BrazilUNIFESP, Escola Paulista Med, Dept Biochem, BR-04044020 São Paulo, BrazilUNIFESP, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilUNIFESP, Escola Paulista Med, Dept Biochem, BR-04044020 São Paulo, BrazilWeb of Scienc

Defining the substrate specificity of mouse cathepsin P

Cathepsin P is a recently discovered placental cysteine protease that is structurally related to the more ubiquitously expressed, broad-specificity enzyme, cathepsin L. We studied the substrate specificity requirements of recombinant mouse cathepsin P using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRSSKQ-EDDnp (Abz, ortho-aminobenzoic acid and EDDnp, N-[2,4-dinitroplieiiyl]ethyleiiediamine). Systematic modifications were introduced resulting in five series of peptides to map the S-3 to S-2' subsites of the enzyme. the results indicate that the subsites S-1, S-2, S-1' and S-2', present a clear preference for hydrophobic residues. the specificity requirements of the S, subsite were found to be more restricted, preferring hydrophobic aliphatic amino acids. the S-3 subsite of the enzyme presents a broad specificity, accepting negatively charged (Glu), positively charged (Lys, Arg), and hydrophobic aliphatic or aromatic residues (Val, Phe). for several substrates, the activity of cathepsin P was markedly regulated by kosmotropic salts, particularly Na2SO4. No significant effect on secondary or tertiary structure could be detected by either circular dichroism or size exclusion chromatography, indicating that the salts most probably disrupt unfavorable ionic interactions between the substrate and enzyme active site. A substrate based upon the preferred P-3 to P-2' defined by the screening study, ortho-aminobenzoic-Glu-Ile-Phe-Val-Phe-Lys-Gln-N-(2,4-dinitrophenyl)ethylenediamine (cleaved at the Phe-Val bond) was efficiently hydrolyzed in the absence of high salt. the k(cat)/K-m for this substrate was almost two orders of magnitude higher than that of the original parent compound. These results show that cathepsin P, in contrast to other mammalian cathepsins, has a restricted catalytic specificity. (C) 2004 Elsevier Inc. All rights reserved.UNIFESP, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilUniv Cidade São Paulo, Neurosci Lab, BR-03071000 São Paulo, BrazilAlfred I DuPont Hosp Children, Dept Biomed Res, Wilmington, de 19803 USAUniv Delaware, Dept Biol Sci, Newark, de 19716 USAUNIFESP, Escola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Scienc

A.S.A. Melo et al. 664 The Candida albicans AAA ATPase homologue

is essential for proper morphology, biofilm formation and activity of secreted aspartyl proteinase

Leviserpin: A Serine Peptidase Inhibitor (Serpin) from the Sugarcane Weevil Sphenophorus levis

Serine peptidase inhibitors (serpins) form a superfamily of proteins covering abroad spectrum of different biological functions. Here we describe the inhibitory characterization of leviserpin, the first serpin from the sugar cane weevil Sphenophorus levis. Leviserpin was able to inhibit bovine trypsin by the formation of the covalent complex serpin-peptidase, demonstrated by SDS-PAGE and mass spectroscopy analysis. We also have determined the cleavage site at the reactive center loop, by the analysis of the polypeptides released from de C-terminus of leviserpin. Moreover we investigated the mRNA expression of leviserpin in different stages of S. levis development. Thus the specificity of leviserpin, in addition with its mRNA coding being transcribed through all lifecycle of the insect, can suggest a possible role in defense mechanism by regulating the action of prophenoloxidase (proPO) activating enzyme.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fed Univ ABC, Ctr Nat & Human Sci, Biol Chem Lab, BR-09210170 Santo Andre, SP, BrazilUniv Fed Sao Carlos, Mol Biol Lab, Dept Genet & Evolut, BR-13565905 Sao Carlos, SP, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilFAPESP: 98/14138-2FAPESP: 06/53607-6CNPq: 312701/2009-8Web of Scienc

The tick-derived rBmTI-A protease inhibitor attenuates the histological and functional changes induced by cigarette smoke exposure

Introduction. Smoking is the main risk factor for chronic obstructive pulmonary disease development and cigarette smoke (CS) exposure is considered an important approach to reproduce in rodents this human disease. We have previously shown that in an elastase-induced model of emphysema, the administration of a protease inhibitor (rBmTI-A) prevented and attenuated tissue destruction in mice. Thus, in this study we aimed to verify the effects of rBmTI-A administration on the physiopathological mechanisms of CS-induced emphysema. Methods. Mice (C57BL/6) were exposed to CS or room air for 12 weeks. In this period, 3 nasal instillations of rBmTI-A inhibitor or its vehicle were performed. After euthanasia, respiratory mechanics were evaluated and lungs removed for analysis of mean linear intercept, volume proportion of collagen and elastic fibers, density of polymorphonuclear cells, macrophages, and density of positive cells for MMP-12, MMP-9, TIMP-1 and gp91phox. Results. The rBmTI-A administration improved tissue elastance, decreased alveolar enlargement and collagen fibers accumulation to control levels and attenuated elastic fibers accumulation in animals exposed to CS. There was an increase of MMP12, MMP-9 and macrophages in CS groups and the rBmTIA only decreased the number of MMP-12 positive cells. Also, we demonstrated an increase in gp91phox in CS treated group and in TIMP-1 levels in both rBmTI-A treated groups. Conclusion. In summary, the rBmTI-A administration attenuated emphysema development by an increase of gp91phox and TIMP-1, accompanied by a decrease in MMP-12 levels